STAT4 regulates the CD8+ regulatory T Cell/T follicular helper cell axis and promotes atherogenesis in insulin-resistant Ldlr-/- Mice

The metabolic syndrome and diabetic conditions support atherosclerosis, but the exact mechanisms for accelerated atherogenesis remain unclear. Although the proinflammatory role of STAT4 in atherosclerosis and diet-induced insulin resistance (IR) was recently established, the impact of STAT4 on atherogenesis in conditions of IR is not known. In this study, we generated Stat4 −/− Ldlr −/− mice that were fed a diabetogenic diet with added cholesterol (DDC). DDC-fed Stat4 −/− Ldlr −/− mice demonstrated improved glucose tolerance, insulin sensitivity, and a 36% reduction in atherosclerosis compared with Ldlr −/− controls. Interestingly, we detected a reduction in T follicular helper (Tfh) cells and plasma B cells but a sharp elevation in CD8 + regulatory T cells (Tregs) in spleens and aortas of Stat4 −/− Ldlr −/− mice compared with Ldlr −/− mice. Similarly, STAT4 deficiency supported CD8 + Treg differentiation in vitro. STAT4-deficient CD8 + Tregs suppressed Tfh cell and germinal center B cell development upon immunization with keyhole limpet hemocyanin, indicating an important role for STAT4 in CD8 + Treg functions in vivo. Furthermore, adoptive transfer of Stat4 −/− Ldlr −/− CD8 + Tregs versus Ldlr −/− CD8 + Tregs resulted in a significant reduction in plaque burden and suppression of Tfh cell and germinal center B cells in DDC-fed Ldlr −/− recipients. STAT4 expression in macrophages (MΦs) also affected the Tfh/CD8 + Treg axis, because conditioned media from Stat4 −/− Ldlr −/− MΦs supported CD8 + Treg differentiation, but not Tfh cell differentiation, in a TGF-β–dependent manner. These findings suggest a novel mechanism by which STAT4 supports atherosclerosis in IR Ldlr −/− mice via STAT4-dependent MΦs, as well as cell-intrinsic suppression of CD8 + Treg generation and functions and maintenance of Tfh cell generation and the accompanying humoral immune response.